Breast cancer is the leading malignancy in women, accounting for more than 350,000 deaths per year in western countries. On the molecular level, about 20% of breast cancers are characterized amplification of the gene encoding the HER2 growth factor receptor (Pauletti, G., et al. (1996), Oncogene, 13: 63-72). Amplification of the HER2 gene results in strong over-expression of the receptor protein and, in turn, confers a growth advantage to the tumor cells. Herceptin, a monoclonal antibody targeting the HER2 protein, improves patient survival even in metastatic breast cancers and is regarded as a paradigm for the potential of a new generation of gene specific drugs (Pegram, M. D., et al. (2004), J Natl Cancer Inst, 96: 759-769).
Amplification of genomic DNA is the result of a selection process aiming at facilitating tumor cell growth, e.g. by high level overexpression of genes that otherwise would be growth rate limiting. Amplified genes, therefore, are likely to be vitally important for tumor cells and represent particular attractive targets for new gene specific therapies. In breast cancer, more than 30 regions of amplification have been detected by means of classical comparative genomic hybridization (CGH), see for example, O'Connell, et al. (2003), Breast Cancer Res Treat, 78: 347-357. Numerous important oncogenes have been identified within these amplicons, for example CMYC at chromosome 8q24, EGFR at 7p21, or CCND1 at 11q13. However, it is assumed that the majority of genes which undergo amplification in breast malignancies has not yet been identified. Thus, there is a hope that other amplified genes can be used in diagnosis, estimation of prognosis and treatment of these diseases.
It has been surprisingly found in the course of the present invention that amplification of the ESR1 gene located at 6q25.1 and encoding the alpha isoform of the estrogen receptor appears to be the most frequent gene amplification that is detectable in breast cancer. In the experiments conducted by the inventors, amplification of the ESR1 gene was observed in 31% of the examined tumors. Even more importantly, the present invention provides evidence that amplification of the ESR1 gene is correlated to an enhanced susceptibility of a tumor, such as a breast cancer, to anti-estrogen treatment, e.g. by administration of Tamoxifen. As a consequence, detection of ESR1 amplification is of significant clinical relevance and may be used in diagnosis and estimation of prognosis and also as a tool for making decisions as to the specific treatment protocol to be used with a particular patient suffering from a proliferative breast disease such as breast cancer.
In view of the enormous number of publications dealing with the estrogen receptors and their relationship with breast cancer, it is surprising that ESR1 amplification in breast cancer has not been identified so far. The inability to detect the 6q25.1 amplicon in several hundreds of breast cancer samples analyzed by classical metaphase CGH during the last decade argues for a generally small amplicon size overstraining the classical CGH resolution. Because the experimental noise is often considerable in array hybridization experiments, single spot peaks are frequently seen and artifacts are difficult to distinguish from true amplification events. In order to overcome these shortcomings, the experiments of the present invention specifically focused on small single gene amplifications. By fluorescence in situ hybridization using a probe specific for the ESR1 gene, putative ESR1 amplification events previously seen in the CGH assay were confirmed.